Over the years, peptidomimetics have become immensely important for both organic and medicinal chemists, as well as the pharmaceutical industry due to the multitude of biological active peptides discovered and characterized. Peptidomimetics are currently exploited to overcome problems associated with their parent peptides (for review, see, e.g. reference 28). These improvements include increased selectivity, oral bioavailability and prolonging the activity by hindering enzymatic degradation within the organism. Among the class of peptidomimetics are the mimetics of protein secondary structure.
Retention of peptide secondary structure is an important tool in drug research when applied to fixing the active conformation of a protein. Constraining the structure of a peptide in its biologically active conformation increases its activity or binding. Within the realm of secondary structures are protein xcex2-turns, which have attracted the attention of medicinal chemists due its importance in such events as hormone-receptor and peptide-enzyme recognition. The xcex2-turn is a segment composed of four amino acids (i to i+1) that occurs when a peptide strand changes its direction. In areas, such as cellular signal transduction, protein-protein interactions are known to be crucial for proper signaling, and may find application for protein xcex2-turn mimetics.
Cellular signal-transduction pathways that are initiated by transmembrane receptors associating with cytoplasmic protein kinases rely on two small protein domains containing sequences of 50-100 amino acids each. These sequences, referred to as Src homology 2 (SH2) and Src homology 3 (SH3) domains, can fold into modules which interact independently of their surrounding sequences. Because SH2 and SH3 domains are involved in protein-protein interactions in the signal transduction pathway, they represent potential targets for therapeutic drugs.
The nature of the interaction between SH3 domains and proteins has been the subject of recent study. The three-dimensional structure of SH3/peptide complexes have been extensively investigated (5-16). These reports revealed that SH3 domains interact with protein ligands via hydrophobic contacts on the surface of the domain and protein. These domains have preferences for certain sequences of amino acids, as determined by screening against peptide combinatorial libraries. The protein ligands form a polyproline type II (PPII) left-handed xcex1-helix containing a consensus sequence that can be generalized as XPpXP, where X is an aliphatic amino acid and p is preferably proline to maintain the helix.
All reported naturally-occurring SH3 ligands are peptides, although certain non-natural SH3 ligands have been described. A variety of peptidomimetics have been described, some of which mimic the xcex2-turn motif (for a review, see, e.g., reference 28). However, no peptidomimetic has been reported to modulate activity of SH3 domains. In a study by Feng et al., a biased combinatorial library was constructed by functionalizing the N-terminus of a pentapeptide with various non-peptide elements, and the constructs were assayed for binding with Src SH3 domain (17). The nonpeptidic moieties appeared to interact with the specificity pocket of Src SH3 domain, lowering the kd almost 1000-fold from the parent pentapeptide ( greater than 1000 mM to 3.4 mM). However, to date, few if any non-peptidic ligands binding in the hydrophobic pockets of SH3 domains have been discovered.
The xcex2-turn has attracted the attention of medicinal chemists due to its importance in such events as hormone-receptor and peptide-enzyme recognition. Rapid degradation of natural peptide substrate or ligands containing the xcex2-turn have lead to the design of compounds mimicking this secondary structure (19, 24-28, 29). A tricyclic compound which includes a 5,6-spirolactam moiety has been described as a Type II xcex2-turn mimetic (A. M. Khalil et al., 25th National Medicinal Chemistry Symposium, University of Michigan, 1996). The configuration of the compound so prepared is reported to provide a xcex2-turn-type structure, rather than an extended conformation.
This invention pertains to peptide mimetics including peptide xcex2-turn mimetics, to compositions which include the peptide xcex2-turn mimetics of the invention, and to methods for inhibiting protein-protein interactions with SH3 domains.
In one aspect, the invention provides a compound represented by the formula (Formula I): 
in which
R1 is hydrogen, alkyl, aryl, alkylcarbonyl, arylcarbonyl, aminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, alkylsulfonyl, arylsulfonyl, or an amino acyl group (e.g., an amino acid residue or a peptide segment);
R2 is hydrogen, alkyl, aryl, or xe2x80x94C(R2a)(R2b)C(O)R5;
R2a and R2b are each independently hydrogen, alkyl, or aryl; or, R2a and R2b, taken together with the carbon atom to which they are attached, form a 3 to 8 membered carbocyclic or heterocyclic ring;
R3 and R4 are each, independently for each occurrence, hydrogen, halogen, alkyl, amino, hydroxy, alkoxy, cyano, or trifluoromethyl; or R2a and R4, together with the atoms to which they are attached, form a 5 or 6-membered heterocyclic ring;
R5 is hydroxy, alkyl, aryl, amino, alkoxy, aryloxy, xe2x80x94SH, alkylthio, arylthio or an amino acyl group (e.g., an amino acid residue or a peptide segment);
m and n are each independently 1 or 2; or a salt thereof.
In a preferred embodiment, R1 is a tetrapeptide, more preferably Ac-Pro-Arg-Pro-Leu or Ac-Ala-Pro-Ala-Leu. In another embodiment, R1 is an amino acyl residue selected from the group consisting of Boc-Leu and Boc-Val. In still other embodiments, R1 is an aminocarboxy moiety represented by the formula xe2x80x94C(O)NHR7, in which R7 is an aryl moiety or a bulky alkyl group. In preferred embodiments, R2 is a moiety represented by the formula xe2x80x94C(R2a)(R2b)C(O)R5. In preferred embodiments, R2b is hydrogen and R2a is a side-chain moiety of a naturally occurring amino acid. In preferred embodiments, R5 is a tripeptide moiety selected from the group consisting of Lys-Pro-Pro-OH and Ala-Pro-Gly-OH. In preferred embodiments, R3 is hydroxy.
In another embodiment, the invention provides a compound represented by the formula (Formula Ia): 
in which
X is xe2x80x94CH2xe2x80x94, S or O;
R1 is hydrogen, alkyl, aryl, alkylcarbonyl, arylcarbonyl, aminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, alkylsulfonyl, arylsulfonyl, or an amino acyl group;
R2b is hydrogen, alkyl, or aryl;
R3 and R4 are each, independently for each occurrence, hydrogen, halogen, alkyl, amino, hydroxy, alkoxy, cyano, or trifluoromethyl;
R5 is hydroxy, alkyl, aryl, amino, alkoxy, aryloxy, xe2x80x94SH, alkylthio, arylthio or an amino acyl group;
m and p are each, independently, 1 or 2; or a salt thereof;
with the proviso that if X is S, p is 1, and R2b is hydrogen, then the configuration at the carbon atom to which R2b is attached is not the R configuration.
In preferred embodiments, R2b is hydrogen.
In another aspect, the invention provides a method for inhibiting a protein-protein interaction mediated by an SH3 domain. The method includes the step of contacting the SH3 domain with a compound of Formula I, such that a protein-protein interaction mediated by an SH3 domain is inhibited. In preferred embodiments, R1 is a tetrapeptide, more preferably Ac-Pro-Arg-Pro-Leu or Ac-Ala-Pro-Ala-Leu. In preferred embodiments, R1 is an amino acyl residue selected from the group consisting of Boc-Leu and Boc-Val. In other embodiments, R1 is an aminocarboxy moiety represented by the formula xe2x80x94C(O)NHR7, in which R7 is an aryl moiety or a bulky alkyl group. In preferred embodiments, R2 is a moiety represented by the formula xe2x80x94C(R2a)(R2b)C(O)R5. In preferred embodiments, R2b is hydrogen and R2a is a side-chain moiety of a naturally occurring amino acid. In preferred embodiments, R5 is a tripeptide moiety selected from the group consisting of Lys-Pro-Pro-OH and Ala-Pro-Gly-OH.
In another aspect, the invention provides a method for inhibiting growth of a cell. The method includes the step of contacting the cell with an effective amount of a compound of Formula I such that growth of the cell is inhibited. In a preferred embodiment, the compound is a compound of Formula Ia.
In another aspect, the invention provides a method for inhibiting a protein-protein interaction mediated by an SH3 domain. The method includes the step of contacting an SH3 domain with an effective amount of a compound of Formula I, such that a protein-protein interaction mediated by an SH3 domain is inhibited. In a preferred embodiment, the SH3 domain is an SH3 domain of Lyn.
In another aspect, the invention provides a library of compounds of Formula I, more preferably a library of compounds of Formula Ia.
In another aspect, the invention provides a pharmaceutical composition. The pharmaceutical composition includes a compound of Formula I (more preferably a compound of Formula Ia), or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable vehicle.
The present inventors have now investigated various known SH3/peptide complexes to design a template that could be exploited for drug discovery. Surveying the conformations of the protein ligands, it was determined (e.g., by molecular modeling) that bound conformations of the ligands differed from minimized unbound peptides (Table 1).(9, 10, 12). Generally, small peptides exhibit little or no conformational preference. However, the inventors have now recognized that bound ligands often appear to exhibit an extended conformation.
It is believed that the unbound peptide ligand relieves A(1-3) strain from the xcex1Cxe2x80x94H and the N-xcex4CH2 bonds in prolines 1 and 2, respectively, in the XPpXP motif, by rotating about the xcex1Cxe2x80x94C(O) bond. Preferably, a synthetic ligand can incorporate this conformational bias. Free rotation around the xcex1Cxe2x80x94C(O) bond can be locked by inserting a methylene bridge between the Pro-1 xcex1xe2x80x94C and the Pro-2 N, (See diagram below) resulting in a spirolactam structure, e.g., the 5,6-spirolactam structure of the compounds of the invention. The invention is based, at least in part, on the understanding that by exploiting the conformational difference between bound and unbound ligand, compounds containing the inventive spirolactam moiety instead of the Pro-Pro dipeptide of natural SH3 domain ligands (i.e., the Pp portion of the XPpXP sequence) can gain binding affinity from the subsequent loss of flexibility.(18) The spirolactam template of the invention is believed both to mimic the Pp portion of the consensus sequence, and also to resemble a protein xcex2-turn structure, a common motif of biologically active peptides. (19-23). 
The novel 5,6 spirolactams of the invention incorporate a rigid structure around which the N- and C-terminal ends allow for functionalization, and change xe2x80x9cdirectionxe2x80x9d of the ligand, as seen in the xcex2-turn motif.
A 5,6 spirolactam of Formula I incorporates a rigid structure around which positions R1, R2, R3 and R4 can be modified to construct a library of compounds diverse in functionality. As shown in Table 2, the various functionalities at the R1 position can be selected to provide ureas, amides (including, e.g., amino acids and small peptide segments), and sulfonamides at the xe2x80x9cN-terminalxe2x80x9d portion of the spirolactam moiety. The R2 position can be modified during the synthesis of the spirolactam using different amino acids. The R5 substituent can be selected to provide a xe2x80x9cC-terminalxe2x80x9d free acid, esters, or amides. The amides can be prepared, e.g., from coupling with 1xc2x0, 2xc2x0 amines, amino acids, or peptides. Exemplary modifications are shown in Table 2. The N-terminal amine can be unsubstituted (R1=H), or, as shown in Table 2, substituted with a variety of functionalities (R1), including peptides, amino acids (which can be protected amino acids), aminocarbonyl groups (providing a compound with an N-terminal urea functionality), and sulfonyl groups. The R2a functionality can be, e.g., hydrogen or an amino acid side chain (e.g., of alanine, isoleucine, lysine, glutamic acid, phenylalanine or tyrosine). The R5 moiety can be, e.g., a peptide, an amino acid, a hydroxy, alkoxy, or aryl group (providing a C-terminal acid or ester functionality), or a primary or secondary amino group (providing a C-terminal amide functionality).
Thus, as discussed in more detail infra, the novel 5,6-spirolactams of the invention can be functionalized at either the N- or the C-terminus (or both) to construct a library of compounds containing the xcex2-turn element. The resultant libraries are also valuable for the generation of lead compounds targeted at specific SH3 domains and other protein-protein interactions, e.g., as described herein.
The term xe2x80x9calkylxe2x80x9d refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C3-C30 for branched chain), and more preferably 20 or fewer. Likewise, preferred cycloalkyls have from 4-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.
Moreover, the term xe2x80x9calkylxe2x80x9d as used throughout the specification and claims is intended to include both xe2x80x9cunsubstituted alkylsxe2x80x9d and xe2x80x9csubstituted alkylsxe2x80x9d, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. Cycloalkyls can be further substituted, e.g., with the substituents described above. An xe2x80x9caralkylxe2x80x9d moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)).
The term xe2x80x9carylxe2x80x9d as used herein includes 5- and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Aryl groups also include polycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as xe2x80x9caryl heterocyclesxe2x80x9d, xe2x80x9cheteroarylsxe2x80x9d or xe2x80x9cheteroaromaticsxe2x80x9d. The aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings which are not aromatic so as to form a polycycle (e.g., tetralin).
The terms xe2x80x9calkenylxe2x80x9d and xe2x80x9calkynylxe2x80x9d refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
Unless the number of carbons is otherwise specified, xe2x80x9clower alkylxe2x80x9d as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, xe2x80x9clower alkenylxe2x80x9d and xe2x80x9clower alkynylxe2x80x9d have similar chain lengths. Preferred alkyl groups are lower alkyls.
The terms xe2x80x9cheterocyclylxe2x80x9d or xe2x80x9cheterocyclic groupxe2x80x9d refer to 3- to 10-membered ring structures, more preferably 4- to 7-membered rings, which ring structures include one to four heteroatoms. Heterocyclyl groups include pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, lactones, cyclic anhydrides, sultams, sultones, and the like. The heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulflhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.
The terms xe2x80x9cpolycyclylxe2x80x9d or xe2x80x9cpolycyclic groupxe2x80x9d refer to two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are xe2x80x9cfused ringsxe2x80x9d. Rings that are joined through non-adjacent atoms are termed xe2x80x9cbridgedxe2x80x9d rings. Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkyl, aralkyl, or an aromatic or heteroaromatic moiety.
The term xe2x80x9cheteroatomxe2x80x9d as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.
The term xe2x80x9caminoxe2x80x9d as used herein, refers to a moiety represented by the formula xe2x80x94NRaRb, in which Ra and Rb are each independently hydrogen, alkyl, aryl, or heterocyclyl, or Ra and Rb together with the nitrogen atom to which they are attached, form a 3- to 8-membered heterocyclic ring.
The term xe2x80x9clinker group,xe2x80x9d as used herein, refers to a linking or spacing moiety which can be used to covalently or non-covalently link a compound to a solid support. Linker groups suitable for use in the invention are known in the art for use in solid-phase synthesis. It will be appreciated by the skilled artisan that a linker group can be a direct bond to a solid support, such a resin bead.
The term xe2x80x9camino acid,xe2x80x9d as used herein, refers to naturally-occurring amino acids, as well as to analogs, derivatives, and mimetics thereof. The SH3 domain inhibitor compounds of the present invention can also comprise peptide analogs, peptide derivatives (e.g.,), and peptidomimetics. As used herein, a xe2x80x9cderivativexe2x80x9d of a compound X (e.g., a peptide) refers to a form of X in which one or more reaction groups on the compound have been derivatized with a substituent group (e.g., alkylated or acylated peptides). As used herein an xe2x80x9canalogxe2x80x9d of a compound X refers to a compound which retains chemical structures of X necessary for functional activity of X yet which also contains certain chemical structures which differ from X. An examples of an analog of a naturally-occurring peptide is a peptide which includes one or more non-naturally-occurring amino acids. As used herein, a xe2x80x9cmimeticxe2x80x9d of a compound X refers to a compound in which chemical structures of X necessary for functional activity of X have been replaced with other chemical structures which mimic the conformation and function of X. Examples of peptidomimetics include peptidic compounds in which the peptide backbone is substituted with one or more benzodiazepine molecules (see e.g., James, G. L. et al. (1993) Science 260:1937-1942) and xe2x80x9cretro-inversoxe2x80x9d peptides (see U.S. Pat. No. 4,522,752 by Sisto), described further below. A xe2x80x9cresiduexe2x80x9d refers to an amino acid or amino acid mimetic incorporated in the peptide compound by an amide bond or amide bond mimetic. Approaches to designing peptide derivatives, analogs and mimetics are known in the art. For example, see Farmer, P. S. in Drug Design (E. J. Ariens, ed.) Academic Press, New York, 1980, vol. 10, pp. 119-143; Ball. J. B. and Alewood, P. F. (1990) J. Mol. Recognition 3:55; Morgan, B. A. and Gainor, J. A. (1989) Ann. Rep. Med. Chem. 24:243; and Freidinger, R. M. (1989) Trends Pharmacol. Sci. 10:270.
An xe2x80x9camino acid mimeticxe2x80x9d refers to a moiety, other than a naturally occurring amino acid, that conformationally and functionally serves as a substitute for a particular amino acid in a peptide-containing compound without adversely interfering to a significant extent with the function of the compound (e.g., binding to an SH3 domain). In some circumstances, substitution with an amino acid mimetic may actually enhance properties of the compound (e.g., interaction of the compound with the SH3 domain). Examples of amino acid mimetics include D-amino acids. Peptides substituted with one or more D-amino acids may be made using well known peptide synthesis procedures. The effect of amino acid substitutions with D-amino acids and other peptidomimetics can be tested using assays as described infra.
The peptide analogs or mimetics of the invention include isosteres. The term xe2x80x9cisosterexe2x80x9d as used herein refers to a sequence of two or more residues that can be substituted for a second sequence because the steric conformation of the first sequence fits a binding site specific for the second sequence. The term specifically includes peptide back-bone modifications (i.e., amide bond mimetics) well known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the xcex1-carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks. Several peptide backbone modifications are known, including "psgr"[CH2S], "psgr"[CH2NH], "psgr"[C(S)NH2], "psgr"[NHCO], "psgr"[C(O)CH2], and "psgr"[(E) or (Z) CHxe2x95x90CH]. In the nomenclature used above, "psgr" indicates the absence of an amide bond. The structure that replaces the amide group is specified within the brackets. Other examples of isosteres include peptides substituted with one or more benzodiazepine molecules (see e.g., James, G. L. et al. (1993) Science 260:1937-1942).
Other possible modifications include an N-alkyl (or aryl) substitution ("psgr"[CONR]), backbone crosslinking to construct lactams and other cyclic structures, or retro-inverso amino acid incorporation ("psgr"[NHCO]). By xe2x80x9cinversoxe2x80x9d is meant replacing L-amino acids of a sequence with D-amino acids, and by xe2x80x9cretro-inversoxe2x80x9d or xe2x80x9cenantio-retroxe2x80x9d is meant reversing the sequence of the amino acids (xe2x80x9cretroxe2x80x9d) and replacing the L-amino acids with D-amino acids. For example, if the parent peptide is Thr-Ala-Tyr, the retro modified form is Tyr-Ala-Thr, the inverso form is thr-ala-tyr, and the retro-inverso form is tyr-ala-thr (lower case letters refer to D-amino acids). Compared to the parent peptide, a retro-inverso peptide has a reversed backbone while retaining substantially the original spatial conformation of the side chains, resulting in a retro-inverso isomer with a topology that closely resembles the parent peptide and is able to bind the selected SH3 domain. See Goodman et al. xe2x80x9cPerspectives in Peptide Chemistryxe2x80x9d pp. 283-294 (1981). See also U.S. Pat. No. 4,522,752 by Sisto for further description of xe2x80x9cretro-inversoxe2x80x9d peptides.
In addition to amino acid-substituted inhibitor compounds, the invention also encompasses SH3 domain inhibitor compounds having other modifications. For example, the amino-terminus of an amino acid or peptide substituent can be modified. The term xe2x80x9camino-derivative groupxe2x80x9d is intended to include amino-terminal modifications of the peptide-containing compounds of the invention. Examples of N-terminal modifications include alkyl, cycloalkyl, aryl, araalkyl, and acyl groups. A preferred N-terminal modification is acylation. Preferred N-terminal acyl or acyloxy groups include acetyl (denoted Ac), and benzoyl, as well as acyloxy groups such as methoxycarbonyl, t-butoxycarbonyl and benzyloxycarbonyl (also denoted xe2x80x9cCbzxe2x80x9d). The N-terminal residue may be linked to a variety of moieties other than amino acids such as polyethylene glycols (such as tetraethylene glycol carboxylic acid monomethyl ether), pyroglutamic acid, succinoyl, methoxy succinoyl, benzoyl, phenylacetyl, 2-, 3-, or 4-pyridylalkanoyl, aroyl, alkanoyl (including acetyl and cycloalkanoyl e.g., cyclohexylpropanoyl), arylalkanoyl, arylaminocarbonyl, alkylaminocarbonyl, cycloalkylaminocarbonyl, alkyloxycarbonyl (carbamate caps), and cycloalkoxycarbonyl, among others.
Except as otherwise noted, standard abbreviations are used throughout this disclosure when describing peptide substituents of the compounds of the invention.
The term xe2x80x9csubstantially pure,xe2x80x9d as used herein, refers to a compound which is substantially free of impurities, including (but not limited to) starting materials, side products, and the like. A compound is xe2x80x9csubstantially purexe2x80x9d if it comprises at least about 80%, more preferably 90%, still more preferably at least about 95% of the composition. If a single isomer of a compound is desired (e.g., a single diastereomer, enantiomer, or regioisomer), the compound is preferably substantially free of any undesired isomers (e.g., the unwanted enantiomer, diastereomers, or regioisomers), i.e., the desired isomer comprises at least about 80%, more preferably 90%, still more preferably at least about 95% of the weight of the isomers present in the composition.
The term xe2x80x9csolid support,xe2x80x9d as used herein, refers to a solid or insoluble phase which can serve as a support or xe2x80x9cscaffoldxe2x80x9d for the preparation of functionalized compounds by solid-phase chemistry. Many solid supports are known in the art and some are commercially available. Exemplary solid supports include cross-linked divinylbenzene-styrene (polystyrene), controlled pore glass (CPG), polyacrylamides, poly(ethyleneglycol)monomethyl ether and poly(ethylene glycol) (PEG), silica gel, cellulose, acrylic acid grafted polypropylene, and the like. Commercially-available solid supports include Wang resin, Merrifield resin, Tentagel, Rapp resin, and the like.
The term xe2x80x9csubjectxe2x80x9d, as used herein, refers to an animal, more preferably a warm-blooded animal, most preferably a mammal, including cattle, sheep, pigs, horses, dogs, cats, rats, mice, and humans.
The term xe2x80x9cSH3 domainxe2x80x9d is art-recognized, and, as used herein, refers to a protein domain involved in protein-protein interactions, such as a domain of a Src tyrosine kinase that negatively regulates kinase activity. Several SH3 domains have been reported and are typically associated with a regulatory function. The invention contemplates modulation of activity, such as activity dependent upon protein-protein interactions, mediated by SH3 domains of proteins (e.g., tyrosine kinases such as src, or proteins involved with transmission of a tyrosine kinase signal, such as Grb2) from organisms including mammals, including humans. For examples of SH3 domains, see the discussion and references cited supra and, e.g., Smithgall, T. E. J. Pharmacol. Toxicol. Methods 1995 34:125-132).
The language xe2x80x9cmodulating an activity mediated by an SH3 domainxe2x80x9d as used herein, refers to inhibiting, abolishing or increasing the activity of a cell-signalling pathway mediated by a protein including an SH3 domain, e.g., by disrupting protein-protein interactions mediated by SH3 domains. In a preferred embodiment, an activity mediated by an SH3 domain is inhibited, for example, an interaction of GRB2 and SOS is inhibited.
It will be noted that the structure of some of the compounds of this invention include asymmetric carbon atoms. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis. Furthermore, alkenes can include either the E- or Z-geometry, where appropriate.
In one aspect, the invention provides spirocyclic compounds useful as ligands for, and modulators of interactions mediated by, SH3 domains.
In one embodiment, the invention provides a compound represented by the formula (Formula I): 
in which
R1 is hydrogen, alkyl, aryl, alkylcarbonyl, arylcarbonyl, aminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, alkylsulfonyl, arylsulfonyl, or an amino acyl group (including, e.g., an amino acid residue or a peptide segment);
R2 is hydrogen, alkyl, aryl, or xe2x80x94C(R2a)(R2b)C(O)R5;
R2a and R2b are each independently hydrogen, alkyl, or aryl; or, R2a and R2b, taken together with the carbon atom to which they are attached, form a 3 to 8 membered carbocyclic or heterocyclic ring;
R3 and R4 are each, independently for each occurrence, hydrogen, halogen, alkyl, amino, hydroxy, alkoxy, cyano, or trifluoromethyl; or R2a and R4, together with the atoms to which they are attached, form a 5 or 6-membered heterocyclic ring;
R5 is hydroxy, alkyl, aryl, amino, alkoxy, aryloxy, xe2x80x94SH, alkylthio, arylthio or an amino acyl group (e.g., an amino acid residue or a peptide segment);
m and n are each independently 1 or 2; or a salt thereof.
In a preferred embodiment, R1 is an amino acyl residue. Thus, for example, R1 can be an amino acid residue, or a polypeptide. In a preferred embodiment, R1 is a peptide, preferably a tetrapeptide. In a particularly preferred embodiment, the tetrapeptide has the formula Ac-Pro-Arg-Pro-Leu or Ac-Ala-Pro-Ala-Leu. In other embodiments, R1 is an amino acyl residue such as Boc-Leu or Boc-Val. In still other embodiments, R1 is an aminocarboxy moiety represented by the formula xe2x80x94C(O)NHR7, in which R7 is preferably an aryl moiety or a bulky alkyl group, e.g., a branched alkyl group. In still another embodiment, R1 is an alkylsulfonyl or arylsulfonyl moiety.
In a preferred embodiment, R2 is a moiety represented by the formula xe2x80x94C(R2a)(R2b)C(O)R5. In a preferred embodiment, R2b is hydrogen and R2a is a side-chain moiety of a naturally occurring amino acid., e.g., a methyl group, an isobutyl group, a phenylmethyl group, and the like. In preferred embodiments, R5 is an amino acyl moiety, more preferably a peptide or polypeptide moiety. In a preferred embodiment, R5 is a tetrapeptide moiety, most preferably Lys-Pro-Pro-OH or Ala-Pro-Gly-OH. In certain embodiments, R5 is hydroxy, alkoxy, or aryloxy. In another embodiment, R5 is an amino group, preferably a monoalkylamino group in which the alkyl group is branched or substituted with one or more aryl moieties. In a preferred embodiment, both R3 and R4 are hydrogen for all occurrences. However, in certain embodiments, R3 is hydroxyl.
In a preferred embodiment, the compound of Formula I has the S configuration at the spirocyclic carbon atom. In other embodiments, the compound of Formula I has the R configuration at the spirocyclic carbon atom. In a preferred embodiment, neither R2a nor R2b is joined with R4 to form a ring structure.
In a preferred embodiment, R2 is a moiety represented by the formula xe2x80x94C(R2a)(R2b)C(O)R5, and R2a and an R4 moiety, together with the atoms to which they are attached, form a 5 or 6-membered heterocyclic ring, with the proviso that if X is S, p is 1, and R2b is hydrogen, then the configuration at the carbon atom to which R2b is attached (the carbon labeled xcex12), is not the R configuration (i.e., the configuration of D-cysteine). In one particularly preferred embodiment, a compound of the invention can be represented by the formula (Formula Ia): 
in which m, R1, R2b, R3, R4 and R5 are as described above, X is xe2x80x94CH2xe2x80x94, S, or O, and p is 1 or 2.
In a preferred embodiment, R1 is an amino acyl residue. Thus, for example, R1 can be an amino acid residue, or a polypeptide. In a preferred embodiment, R1 is a peptide, preferably a tetrapeptide. In a particularly preferred embodiment, the tetrapeptide has the formula Ac-Pro-Arg-Pro-Leu or Ac-Ala-Pro-Ala-Leu. In other embodiments, R1 is an amino acyl residue such as Boc-Leu or Boc-Val. In still other embodiments, R1 is an aminocarboxy moiety represented by the formula xe2x80x94C(O)NHR7, in which R7 is preferably an aryl moiety or a bulky alkyl group, e.g., a branched alkyl group. In still another embodiment, R1 is an alkylsulfonyl or arylsulfonyl moiety.
In a preferred embodiment, of the compound of Formula Ia, the configuration at the two ring junction chiral carbons (labeled as xcex11 and xcex12) is the same, i.e., xcex11 and xcex12 are both R or both S in configuration, more preferably both S. In a preferred embodiment, R2b is hydrogen. In preferred embodiments, R5 is an amino acyl moiety, more preferably a peptide or polypeptide moiety. In a preferred embodiment, R5 is a tripeptide moiety, most preferably Lys-Pro-Pro-OH or Ala-Pro-Gly-OH. In certain embodiments, R5 is hydroxy, alkoxy, or aryloxy. In another embodiment, R5 is an amino group, preferably a monoalkylamino group in which the alkyl group is branched or substituted with one or more aryl moieties.
In some cases, compounds having fewer amino acid residues are more easily and economically prepared, and are more stable, than compounds which include more amino acid residues. Thus, in preferred embodiments, a compound of Formula I comprises no more than about 7 amino acid residues (e.g., R1 and R2, or R1 and R5, together do not include more than about 7 amino acid residues), more preferably not more than about 5 amino acid residues, and still more preferably not more than about 3 amino acid residues.
The compounds of the invention can be prepared by the methods described herein, or by other methods which will be routine for the ordinarily skilled artisan in light of the teachings herein. For example, the spirocyclic compounds of the invention can be prepared from chiral or non-chiral starting materials, and can be enantiomerically pure or a racemic mixture of compounds. Although the synthesis described in Example 1 produces a racemic modification (e.g., racemization occurs at the alkylation step), enantiomeric purity can be retained through the alkylation step by means of xe2x80x9cself-reproductionxe2x80x9d of chirality (see, e.g., Seebach, D. et al. J. Am. Chem. Soc. (1983) 105:5390-5398). Alternatively, chiral auxiliaries or catalysts can be employed to obtain enantiomerically enriched products. Enantiomers can also be separated by resolution techniques, such as chiral chromatography or the use of chiral acids or bases, as are conventional in the art.
Furthermore, analogs and derivatives of the compounds of the invention can be prepared by standard synthetic methodologies in light of the teachings herein. Thus, for example, substitution of an appropriately-protected 3- or 4-hydroxyproline (3- and 4-hydroxyproline are available, e.g., from Sigma Chemical Co., St. Louis, Mo.) in place of proline in the synthesis described in Example 1, provides compounds of the invention, e.g., in which R3 is a hydroxy group (or a derivative thereof) and m is one.
It will be appreciated by the skilled artisan that the conformation of the spirolactam peptidomimetics of the invention can be altered by appropriate selection of substituents and configuration (e.g., chiral configuration) of the substituents selected. The configuration of the ring junction carbons can also affect the conformation of the peptidomimetics of the invention. Thus, for example, in compounds of Formula Ia, the configuration of the carbon atom to which the xe2x80x94C(O)R5 moiety is attached (the carbon atom labeled as xcex12 in Formula Ia), can affect the conformation of the peptidomimetic; one epimer at the xcex12 carbon can assume the xcex2-turn structure, while the other epimer at the xcex12 carbon can assume an extended conformation, i.e., a conformation which may be useful as an SH3 domain modulator. In a preferred embodiment, a compound of the invention has an extended conformation and/or is capable of modulating protein-protein interactions mediated by an SH3 domain.
In another aspect, the invention provides libraries of compounds of Formula I. Libraries of the invention are useful, e.g., for drug discovery. For example, a library of the invention can be screened (e.g., according to the methods described herein) to determine whether the library includes compounds having a pre-selected activity. Thus, for example, a library can be screened to determine whether compounds of the library have SH3 domain binding activity or any other activity which can be detected in vitro or in vivo, e.g., anti-inflammatory activity, cell growth stimulatory activity, anti-neoplastic activity, and the like.
The synthesis of combinatorial libraries is well known in the art and has been reviewed (see, e.g., E. M. Gordon et al., J. Med. Chem. 37:1385-1401 (1994)). Thus, the subject invention contemplates methods for synthesis of combinatorial libraries of compounds of Formula I. Such libraries can be synthesized according to a variety of methods. For example, a xe2x80x9csplit-poolxe2x80x9d strategy can be implemented in the following way: beads of a functionalized polymeric support (such as Wang resin) are placed in a plurality of reaction vessels. To each aliquot of beads is added a solution of an N-Boc protected amino acid, and a coupling reagent (e.g., DCC) is added to each vessel. The reactions are allowed to proceed to yield a plurality of immobilized amino acids. The aliquots of derivatized beads are then washed, xe2x80x9cpooledxe2x80x9d (i.e., recombined), the N-Boc protecting group is removed (e.g., with a solution of TFA in DCM) and the pool of beads is again divided, with each aliquot being placed in a separate reaction vessel. To each reaction vessel is added a solution of an aldehyde (e.g., an aldehyde such as compound 6 of Scheme 1) and a reducing agent such as sodium cyanoborohydride, and reductive amination occurs to yield a plurality of reaction vessels each containing a plurality of compounds immobilized on solid support. The immobilized compounds can be cyclized, e.g., as shown in Scheme 1, by deprotection and intramolecular lactamization, to provide a library of compounds of Formula I (in which R2 is xe2x80x94C(R2a)(R2b)C(O)R5). The library of immobilized compounds can then be washed to remove impurities. In certain embodiments, the library of compounds can be further treated, e.g., by deprotection and derivatization of the amino functionality. Further processing can optionally include cleavage from the solid support, if desired, and further reaction (e.g., substitution or further functionalization).
In another illustrative method of combinatorial synthesis, a xe2x80x9cdiversomer libraryxe2x80x9d is created by a modification of the method of Hobbs DeWitt et al. (Proc. Natl. Acad. Sci. U.S.A. 90:6909 (1993)). Other synthesis methods, including the xe2x80x9ctea-bagxe2x80x9d technique of Houghten (see, e.g., Houghten et al., Nature 354:84-86 (1991)) can also be used to synthesize libraries of compounds according to the subject invention.
For examples of combinatorial library synthesis, see Examples 6 and 9, infra.
Combinatorial libraries can be screened to determine whether any members of the library have a desired activity, and, if so, to identify the active species. Methods of screening combinatorial libraries have been described (see, e.g., Gordon et al., J. Med. Chem., op. cit.). Soluble compound libraries can be screened by affinity chromatography with an appropriate receptor to isolate ligands for the receptor, followed by identification of the isolated ligands by conventional techniques (e.g., mass spectrometry, NMR, and the like). Immobilized compounds can be screened by contacting the compounds with a soluble receptor; preferably, the soluble receptor is conjugated to a label (e.g., fluorophores, colorimetric enzymes, radioisotopes, luminescent compounds, and the like) that can be detected to indicate ligand binding. Alternatively, immobilized compounds can be selectively released and allowed to diffuse through a membrane to interact with a receptor. Exemplary assay formats useful for screening the libraries of the invention are known in the art (see, e.g., E. M. Gordon et al., J. Med. Chem. 37:1385-1401 (1994)). See also Example 12, infra.
Combinatorial libraries of compounds can also be synthesized with xe2x80x9ctagsxe2x80x9d to encode the identity of each member of the library (see, e.g., W. C. Still et al., U.S. Pat. No. 5,565,324 and PCT Publication No. WO 94/08051). In general, this method features the use of inert, but readily detectable, tags, that are attached to the solid support or to the compounds. When an active compound is detected (e.g., by one of the techniques described above), the identity of the compound is determined by identification of the unique accompanying tag. This tagging method permits the synthesis of large libraries of compounds which can be identified at very low levels.
In preferred embodiments, the libraries of compounds of the invention contain at least 2 compounds, more preferably at least about 30 compounds, more preferably at least about 100 compounds, more preferably at least about 500 compounds, and still more preferably at least about 1000 compounds. In certain embodiments, the libraries of the invention can include at lest 104 compounds, 105 compounds, 106 compounds, or 107 compounds. However, in certain preferred embodiments, the libraries of compounds of the invention contain fewer than 109 compounds, more preferably fewer than 108 compounds, and still more preferably fewer than 107 compounds.
A library of compounds is preferably substantially pure, i.e., substantially free of compounds other than the intended products, e.g., members of the library. In preferred embodiments, the purity of a library produced according to the methods of the invention is at least about 50%, more preferably at least about 70%, still more preferably at least about 90%, and most preferably at least about 95%.
The libraries of the invention can be prepared according to the methods described herein, or by other synthetic methods which will be apparent to the ordinarily-skilled artisan. In general, such methods will involve the preparation of libraries by providing variegated populations of amino acids or of the 5,6-spirolactams of the invention. The term xe2x80x9cvariegated populationxe2x80x9d, as used herein, refers to a population including at least two different chemical entities, e.g., of different chemical structure. For example, a xe2x80x9cvariegated populationxe2x80x9d of amino acids would comprise at least two different amino acids. Similarly, a variegated population of spirolactams comprises at least two different spirolactams.
In another aspect, the invention provides pharmaceutical compositions comprising a compound of the invention, or a pharmaceutically-acceptable salt thereof, and a pharmaceutically-acceptable carrier.
The pharmaceutical compositions of the invention comprise a therapeutically-effective amount of one or more of the compounds described above (e.g., Compounds of Formula I, including Formula Ia), formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; or (4) intravaginally or intrarectally, for example, as a pessary, cream or foam.
The phrase xe2x80x9ctherapeutically-effective amountxe2x80x9d as used herein means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect, e.g., treating (i.e., preventing or ameliorating) cancer in a subject, or inhibiting protein-protein interactions mediated by an SH3 domain in a subject, at a reasonable benefit/risk ratio applicable to any medical treatment.
The phrase xe2x80x9cpharmaceutically acceptablexe2x80x9d is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The phrase xe2x80x9cpharmaceutically-acceptable carrierxe2x80x9d as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer""s solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.
As set out above, certain embodiments of the present compounds can contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable acids. The term xe2x80x9cpharmaceutically-acceptable saltsxe2x80x9d in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, e.g., Berge et al. (1977) xe2x80x9cPharmaceutical Saltsxe2x80x9d, J. Pharm. Sci. 66:1-19).
In other cases, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. The term xe2x80x9cpharmaceutically-acceptable saltsxe2x80x9d in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like (See, for example, Berge et al., supra).
Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
Formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be administered as a bolus, electuary or paste.
In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the compound of the invention in the proper medium. Absorption enhancers can also be used to increase the flux of the compound of the invention across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound of the invention in a polymer matrix or gel.
Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.
Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
When the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
The preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral or topical administration is preferred.
The phrases xe2x80x9cparenteral administrationxe2x80x9d and xe2x80x9cadministered parenterallyxe2x80x9d as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
The phrases xe2x80x9csystemic administration,xe2x80x9d xe2x80x9cadministered systemically,xe2x80x9d xe2x80x9cperipheral administrationxe2x80x9d and xe2x80x9cadministered peripherallyxe2x80x9d as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient""s system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the derivative (e.g., ester, salt or amide) thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
In general, a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, doses of the compounds of this invention for a patient, when used for the indicated effects, will range from about 0.0001 to about 100 mg per kilogram of body weight per day, more preferably from about 0.01 to about 50 mg per kg per day, and still more preferably from about 0.1 to about 40 mg per kg per day.
If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical composition.
In still another aspect, the invention provides methods for modulating an activity mediated by an SH3 domain. In general, the methods include the step of contacting an SH3 domain with a compound of the invention, such that activity of the SH3 domain is modulated.
The methods of the invention provide means for inhibiting protein-protein interactions mediated by SH3 domains. Proteins with SH3 domains couple protein-tyrosine kinases to signalling networks involved in growth regulation (see, e.g., Smithgall, op cit. and references cited therein). Disruption of growth-regulatory signal transduction can result in inhibition of cell growth. Accordingly, the invention provides methods for inhibiting growth of cells, including microbial cells and transformed cells, e.g., by inhibiting protein-protein interactions mediated by SH3 domains involved in growth-regulatory signal transduction. Thus, the invention provides methods for treating conditions associated with abnormal or undesired cell growth, including, e.g., fungal or bacterial infections, neoplastic conditions (including cancer), and the like.
In one embodiment, the invention provides a method for modulating intracellular signaling pathways by disrupting particular protein-protein interactions mediated by SH3 domains. For instance, the SH3 inhibitors of the present invention can be used to affect the responsiveness of a cell to a growth factor, cytokine or other receptor ligand, and to inhibit the proliferation of transformed cells or to render transformed cells more sensitive to cytostatic or cytotoxic agents. The SH3 target of the subject inhibitors can range from the interaction between, for example, an activated receptor complex and the initial cytoplasmic proteins involved in triggering a particular set of intracellular signaling pathways, to the last SH3-mediated interaction in a specific pathway, such as the formation of a transcription factor complex or allosteric regulation of an enzymatic activity. Thus, the inhibitors of the present invention can be used to inhibit the interaction between an SH3-binding signal transduction protein such as Grb2, and such SH3-containing proteins as, for example, Src, p85, Fyn, Lyn, Hck, Syk, Grb2, Gap, STAT, p47-phox, p67-phox, Btk, and the like.
Interaction with SH3 domains can lead to activation of the biochemical function associated with the target protein. Examples of this phenomenon include the microtubule-associated GTPase, dynamin Gout et al., 1993) as well as PI-3K (Pleiman et al., 1994) In the case of PI-3K, binding of the SH3 domains of the Src-related kinases Fyn and Lyn significantly increased PI-3K enzymatic activity (Pleiman et al., 1994). This event may have biological relevance in B-cells, in which occupation of the B-cell receptor with antigen leads to activation of Lyn and its association with the p85 subunit of PI-3K. Introduction of p85-based prolinie-rich peptides into permeabilized B-cells completely blocked antigen receptor-mediated activation of PI-3K (Pleiman et al., 1994). This result suggests that the Lyn SH3-p85 interaction is required for PI-3K activation in intact cells. Thus, analogs based on the target motif in this case (p85 subunit) can inhibit protein-protein interaction and activation of the downstream effector.
In a preferred embodiment, the methods of the invention for inhibition of protein-protein interactions mediated by SH3 domains include the step of contacting an SH3 domain with a compound of Formula I, more preferably a compound of Formula Ia. In preferred embodiments, the compound is selected to preferentially inhibit an SH3 domain of an abnormal cell (such as a cancer cell), or a pathogen cell (e.g., a fungal pathogen). Thus, in preferred embodiments, the methods of the invention comprise contacting an SH3 domain of a target protein with a compound of the invention which is selective for the target protein SH3 domain.
Compounds useful in the methods of the invention can be determined by the skilled artisan in light of the teaching herein using no more than routine experimentation. For example, example 12, infra, provides an assay for compounds that inhibit an interaction between a natural peptide ligand and an SH3 domain. Thus, the assay of Example 12 provides a rapid, high-throughput screening system for identifying compounds useful for inhibiting protein-protein interactions mediated by SH3 domains. A similar assay, using an SH3 domain of a non-target protein, can be used to determine the selectivity of a given compound of the invention for a target protein in preference to a non-target protein. Other assays which measure the ability of a compound to inhibit proliferation, to alter the responsiveness of a cell to a growth factor, and the like, will be apparent to the ordinarily-skilled artisan. For example, the ability of a compound of the invention to inhibit cell growth in culture can be measured by standard assays.
In the Examples, all temperatures are in degrees Centigrade (xc2x0 C.). Melting points are uncorrected and were determined on a Mel-Temp II (Laboratory Devices, USA) using open capillary tubes. Infrared spectra (IR) were recorded on a Perkin-Elmer Spectrum 1000. Mass spectra (MS) were recorded on a Platform 2 Micromass instrument. Nuclear magnetic resonance (NMR) spectra were measured on a Varian 200 instrument in the specified solvent with tetramethylsilane (TMS) as internal standard for 1H NMR. For 13C NMR spectra, the deuterated solvent peak was used as the reference with its position set relative to TMS.
Where possible all reactions were followed by thin layer chromatography (TLC) and visualized using UV fluorescence, 3% KMnO4 (aqueous) staining, and/or dodecamolybdophosphoric acid. Commercial thin layer and preparative layer chromatography plates were Si250F and Si500F, respectively, from J. T. Baker. Flash chromatography was performed using 40 xcexcm xe2x80x9cBakerxe2x80x9d silica gel from J. T. Baker. All solvent mixtures are listed as volume ratios.
Abbreviations: EDCI: 1-(3-dimethylaminopropyl)-ethylcarbodiimide; EtOAc, ethyl acetate; BOP: benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate; HOBt: 1-hydroxybenzotriazole; NMM: N-methylmorpholine